Combination therapy for treatment of coronary artery disease

ABSTRACT

An effective combined dose of an anti-CD47 agent and an anti-TNFα agent is administered to the subject in a dose and for a period of time effective to stabilize, prevent or reduce atherosclerotic plaque in the individual.

CROSS-REFERENCE

This application is a continuation and claims the benefit of 371application Ser. No. 15/551,881, filed Aug. 17, 2017, and claims thebenefit of PCT Application No. PCT/US2016/019633, filed Feb. 25, 2016,which claims benefit of U.S. Provisional Patent Application No.62/126,260, filed Feb. 27, 2015, which applications are incorporated byreference herein in their entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under contract HL103605awarded by the National Institutes of Health. The Government has certainrights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A sequence Listing is provided herewith a text file,S13-370_STAN-1065US2.Seqlist_ST25.txt, created on Mar. 23, 2016 andhaving a size of 1243 bytes. The contents of the text file areincorporated by reference herein in their entirety

BACKGROUND OF THE INVENTION

Atherosclerotic cardiovascular disease (ASCVD) remains the primary causeof morbidity and mortality worldwide. Patients with ASCVD represent aheterogeneous group of individuals, with a disease that progresses atdifferent rates and in distinctly different patterns. Despiteappropriate evidence-based treatments for patients with ASCVD,recurrence and mortality rates remain 2-4% per year.

In general, atherosclerosis is believed to be a complex diseaseinvolving multiple biological pathways. Variations in the naturalhistory of the atherosclerotic disease process, as well as differentialresponse to risk factors and variations in the individual response totherapy, reflect in part differences in genetic background and theirintricate interactions with the environmental factors that areresponsible for the initiation and modification of the disease.Atherosclerotic disease is also influenced by the complex nature of thecardiovascular system itself where anatomy, function and biology allplay important roles in health as well as disease.

Traditional risk factors account for approximately half of anindividual's lifetime risk of cardiovascular disease. The balance,therefore, is accounted for by a combination of unmeasured environmentalexposures and genetic factors. The recent advent of the genome-wideassociation study (GWAS) platform has made it possible to investigatethe heritable component of complex polygenic disorders, such asatherosclerotic coronary artery disease (CAD). Using this approach, aregion on chromosome 9p21.3 has repeatedly been identified in GWAS asthe top locus for complex cardiovascular disease (Helgadottir et al.(2007) Science 316:1491-1493; McPherson et al. (2007) Science316:1488-1491).

Available data suggest that the risk-associated polymorphisms: 1) arevery common, with as much as a fifth of the world population carryingtwo copies of the risk allele (minor allele frequency ˜50%) (Deloukas etal. (2013) Nat Genet 45:25-33); 2) are independent of all establishedrisk factors, suggesting a novel mechanism of action (Cunnington andKeavney (2011) Curr Atheroscler Rep 13:193-201); 3) are responsible forup to 21% of the attributable risk of myocardial infarction (MI); and 4)promote risk across a spectrum of vascular diseases, including CAD,stroke, peripheral artery disease (PAD) and abdominal aortic aneurysm(AAA) (Helgadottir et al. (2008) Nat Genet 40:217-224).

Atherosclerotic plaque consists of accumulated intracellular andextracellular lipids, smooth muscle cells, connective tissue, andglycosaminoglycans. The earliest detectable lesion of atherosclerosis isthe fatty streak, consisting of lipid-laden foam cells, which aremacrophages that have migrated as monocytes from the circulation intothe subendothelial layer of the intima, which later evolves into thefibrous plaque, consisting of intimal smooth muscle cells surrounded byconnective tissue and intracellular and extracellular lipids.

SUMMARY OF THE INVENTION

Methods are provided for the prevention and treatment of coronary arterydisease (CAD) in a subject, including without limitation methods ofpreventing or treating atherosclerosis. In the methods of the invention,an effective combined dose of an anti-CD47 agent and an anti-TNFα agentare administered to the subject in a dose and for a period of timeeffective to stabilize, prevent or reduce atherosclerotic plaque in theindividual. In some embodiments the combined dosage provides for asynergistic response relative to the response obtained with either theanti-CD47 agent or the anti-TNFα agent delivered as a monotherapy. Insome embodiments the effective dose of the anti-CD47 agent in thecombined therapy is lower than the effective dose required as amonotherapy. In some embodiments the effective dose of the anti-TNFαagent in the combined therapy is lower than the effective dose requiredas a monotherapy.

In some embodiments, the subject is homozygous or heterozygous for a9p21 risk allele. In some such embodiments the methods include genetictesting of the subject for the presence of a 9p21 risk allele. In othersuch embodiments the subject has been previously diagnosed for thepresence of a 9p21 risk allele, where such methods may include, withoutlimitation, analyzing a sample of genomic DNA from the individual forthe presence of sequences of human chromosome 9p21 associated risk ofCAD, including SNPs associated with the risk locus.

Another aspect of the present invention relates to the use of aneffective combined dose of an anti-CD47 agent and an anti-TNFα agent inthe manufacture of a medicament to stabilize, prevent or reduceatherosclerotic plaque, wherein the medicament is administered to anindividual having or at risk of having atherosclerosis.

Still another aspect of the present invention provides a kit tostabilize, prevent or reduce atherosclerotic plaque. The kit includes aneffective combined dose of an anti-CD47 agent and an anti-TNFα agent, inan amount sufficient to stabilize, prevent or reduce atheroscleroticplaque. The kit may also include reagents for genotyping at humanchromosome 9p21, including alleles of rs10757278 and rs1333049. The kitmay also instructions for use, reagents for monitoring atheroscleroticdisease, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1. Atherosclerotic plaque in the thoracic aorta is reduced byanti-CD47 Ab administration. Thoracic aortae from mice treated withanti-CD47 Ab (MIAP410) or control Ab (IgG) were harvested and pinned.Atherosclerotic plaque (white) area was quantified under low powermicroscopy in a blinded fashion. The percentage of the aortic vesselarea covered by atherosclerotic plaque was quantified for each animaland is shown to be significantly reduced in animals which receivedanti-CD47 Ab.

FIG. 2. Representative explanted aortas from control Ab treated mice(IgG) reveal a high incidence of aneurysms after angiotensin infusion.Representative explanted aortas from antiCD47 Ab treated mice (MIAP410)reveal a low incidence of aneurysms after angiotensin infusion. Thegraph depicts data points for anti-CD47 Ab treatment reducing aneurysmsize.

FIG. 3. Staining with DAPI, for cleaved caspase, and Tunel. The graphsillustrate a decrease in the number of apoptotic cells with anti-CD47treatment, as evidenced by staining for cleaved caspase 3. The cleavedcaspase % has a p<0.05. The TUNEL staining is confirmatory.

FIG. 4. The in vivo phagocytic index associated with atherosclerosis isincreased by treatment with anti-CD47, as illustrated by a decrease inthe number of free apoptotic bodies not associated with macrophages.

FIG. 5. CD47 expression is augmented by TNF-α, which blunts thereduction of CD47 expression in smooth muscle cells due to apoptosis.

FIG. 6. Graphs depicting cell surface CD47 expression in HUVEC andmAoSMC in the absence and presence of TNF-α.

FIG. 7. CD47 is upregulated in human atherosclerotic plaque compared tonormal vascular tissue.

FIG. 8. Overexpression of NF-κB subunits, including NFκB1 activateexpression of CD47.

FIG. 9. Expression of CD47, TNFRSF1A and NFκB correlate in humanatherosclerotic carotid endarterectomy samples.

FIG. 10. Expression of CD47 and TNFα correlate in human atheroscleroticcoronary artery samples.

FIG. 11A-11C. (FIG. 11A) Pro-atherosclerotic stimuli such as oxLDL andapoptotic stimuli (STS) induce the phagocytic clearance of smooth musclecells (SMC). The presence of TNF-α blunts this increase in phagocytosis(FIG. 11B). Anti-CD47 Ab is disproportionately effective at promotingphagocytosis even in the presence of TNF-α (FIG. 11C).

FIG. 12. In vitro data showing that anti-CD47 therapy can synergize withanti-TNF-alpha therapy to promote efferocytosis/phagocytosis.Concomitant inhibition of CD47 and TNFα using anti-CD47 antibody andinfliximab, respectively, produces synergistic benefit in the clearanceof diseased vascular cells.

FIG. 13. Mechanistic data showing that TNF inhibitors (SPD 304 andInfliximab) each reduce CD47 expression and synergize with anti-CD47 Abto promote efferocytosis. A. Pretreatment of mouse vascular smoothmuscle cells with a chemical inhibitor (SPD 304) or a monoclonalantibody (infliximab) directed against TNF-α prevents the increase inCD47 expression normally seen after TNFα exposure. B. The combination ofthese agents with anti-CD47 antibody therapy leads to a synergisticincrease in efferocytosis with in vitro phagocytosis assays.***=p<0.001, **=p<0.01, *=p<0.05.

FIG. 14. In vivo data showing that combination therapy (anti-CD47 Abplus the TNF-α inhibitor Etanercept) reduces atherosclerosis to agreater extent that anti-CD47 Ab (MIAP410) alone. Data presented as OilRed O positive atherosclerotic area in the aortic sinus of mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of treating a subject foratherosclerosis, including conditions such as CAD, peripheral arterydisease (PAD) and cerebrovascular disease, by administering an effectivecombined dose of an anti-CD47 agent and an anti-TNFαagent. In someembodiments, the subject is homozygous or heterozygous for a 9p21 riskallele.

Coronary artery disease (CAD): is a narrowing or blockage of thearteries and vessels that provide oxygen and nutrients to the heart. Itis caused by atherosclerosis, an accumulation of fatty materials on theinner linings of arteries. The resulting blockage restricts blood flowto the heart. When the blood flow is completely cut off, the result is aheart attack. CAD is the leading cause of death for both men and womenin the United States.

Atherosclerosis (also referred to as arteriosclerosis, atheromatousvascular disease, arterial occlusive disease) as used herein, refers toa cardiovascular disease characterized by plaque accumulation on vesselwalls and vascular inflammation. The plaque consists of accumulatedintracellular and extracellular lipids, smooth muscle cells, connectivetissue, inflammatory cells, and glycosaminoglycans. Inflammation occursin combination with lipid accumulation in the vessel wall, and vascularinflammation is with the hallmark of atherosclerosis disease process.

Myocardial infarction is an ischemic myocardial necrosis usuallyresulting from abrupt reduction in coronary blood flow to a segment ofmyocardium. In the great majority of patients with acute MI, an acutethrombus, often associated with plaque rupture, occludes the artery thatsupplies the damaged area. Plaque rupture occurs generally in vesselspreviously partially obstructed by an atherosclerotic plaque enriched ininflammatory cells. Altered platelet function induced by endothelialdysfunction and vascular inflammation in the atherosclerotic plaquepresumably contributes to thrombogenesis. Myocardial infarction can beclassified into ST-elevation and non-ST elevation MI (also referred toas unstable angina). In both forms of myocardial infarction, there ismyocardial necrosis. In ST-elevation myocardial infraction there istransmural myocardial injury which leads to ST-elevations onelectrocardiogram. In non-ST elevation myocardial infarction, the injuryis sub-endocardial and is not associated with ST segment elevation onelectrocardiogram. Myocardial infarction (both ST and non-ST elevation)represents an unstable form of atherosclerotic cardiovascular disease.Acute coronary syndrome encompasses all forms of unstable coronaryartery disease. Heart failure can occur as a result of myocardialdysfunction caused by myocardial infraction.

Angina refers to chest pain or discomfort resulting from inadequateblood flow to the heart. Angina can be a symptom of atheroscleroticcardiovascular disease. Angina may be classified as stable, whichfollows a regular chronic pattern of symptoms, unlike the unstable formsof atherosclerotic vascular disease. The pathophysiological basis ofstable atherosclerotic cardiovascular disease is also complicated but isbiologically distinct from the unstable form. Generally stable angina isnot myocardial necrosis.

“Treatment”, “treating”, “treat” and the like are used herein togenerally refer to obtaining a desired pharmacologic and/or physiologiceffect. The effect can be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete stabilization or cure fora disease and/or adverse effect attributable to the disease. “Treatment”as used herein covers any treatment of a disease in a mammal,particularly a human, and includes: (a) preventing the disease orsymptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom. Those in need of treatment include individuals alreadydiagnosed with CAD, e.g. atherosclerosis, as well as those in which thedisease is to be prevented.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.Preferably, the mammal is human.

An “effective amount” is an amount sufficient to effect beneficial ordesired clinical results. An effective amount can be administered in oneor more administrations. For purposes of this invention, an effectiveamount of an anti-CD47 agent combined with an anti-TNFα agent is anamount that is sufficient to palliate, ameliorate, stabilize, reverse,prevent, slow or delay the progression of the disease state, e.g.atherosclerosis or atherosclerotic plaque, by increasing phagocytosis ofa target cell. For example, in an animal model the percent of aorticsurface area with atherosclerotic plaque may be reduced 25%, 50%, 75% ormore relative to a control treated animal. Similar effects may beobtained with indicia appropriate for human patients, including withoutlimitation C-reactive protein [CRP] and fibrinogen;lipoprotein-associated phospholipase A2 [Lp-PLA2] and myeloperoxidase[MPO]; growth differentiation factor-15 [GDF-15]) inflammatory markers;ambulatory arterial stiffness, IVUS imaging, and the like. See, forexample Krintus et al. (2013) Crit Rev Clin Lab Sci. 11:1-17; Kollias etal. (2012) Atherosclerosis 224(2):291-301; and Kollias et al. (2011)Int. J. Cardiovasc. Imaging 27(2):225-37, each herein specificallyincorporated by reference.

Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokineproduced by macrophages and lymphocytes that mediates inflammation in anumber of conditions. The strategies for inhibiting TNF that have beenmost extensively studied to date consist of monoclonal anti-TNFαantibodies, anti-TNF receptor antibodies, and soluble TNF receptors(sTNF-R). Some specific examples of anti-TNF agents that are in currentclinical use are listed below.

Infliximab (IFX) is a recombinant IgG1 monoclonal antibody specific forTNF-α that hinders the cytokine from triggering the cellular TNFreceptor complex. IFX needs to be administered by intravenous infusionand has a terminal half-life of 8-10 days. Conventionally it isadministered every 4-8 weeks and the dosage varies from 3 to 6 (to 10)mg/kg. Combination therapies of the present invention may use a dose ordosing schedule that provides for a lower dose or reduced dosingschedule relative to conventional schedules.

Adalimumab is a monoclonal antibody of recombinant immunoglobulin (IgG1)containing only human sequences of peptides. It is an antagonist ofTNF-α, which is able to prevent the binding of TNF-α to its receptors.It has a half-life of 10-20 days. The conventional dose of ADA is 25 mgs.c. twice a week. Combination therapies of the present invention mayuse a dose or dosing schedule that provides for a lower dose or reduceddosing schedule relative to conventional schedules.

Golimumab is a human anti-TNF-α monoclonal antibody that is generatedand matured in an in vivo system. GOLI has a high affinity andspecificity for human TNF-α and effectively neutralizes TNF-αbioactivity in vitro. Conventional dosing is from 50 to 100 mg.Combination therapies of the present invention may use a dose or dosingschedule that provides for a lower dose or reduced dosing schedulerelative to conventional schedules.

Etanercept is a genetically engineered protein comprising two moleculesof the extracellular domain of TNF receptor II (p75) and the Fc portionof IgG1. Due to its half-life of 3-5.5 days, ETN is conventionallyadministered subcutaneously (s.c), either on a weekly basis (50 mg) ortwice a week (25 mg). Combination therapies of the present invention mayuse a dose or dosing schedule that provides for a lower dose or reduceddosing schedule relative to conventional schedules.

Certolizumab is a pegylated anti-TNF-alpha monoclonal antibody.Certolizumab is a humanized antibody fragment (Fab′) that is attached topolyethylene glycol to allow for less frequent administration.Certolizumab has a high affinity for human TNF-alpha, selectivelytargeting TNF-alpha in inflamed tissue. Although the presence of a Fab′portion allows certolizumab to retain the potency of the entireantibody, certolizumab is unable to bind phagocytic cells or to lysecells because of a lack of an Fc portion. The elimination half-life ofcertolizumab was demonstrated to be 311 hours.

CD47, also known as integrin associated protein (IAP,) is a 50 kDamembrane receptor that has extracellular N-terminal IgV domain, fivetransmembrane domains, and a short C-terminal intracellular tailtransmembrane, belonging to the immunoglobulin superfamily, withinteracts with integrins, most commonly integrin αvβ3, thrombospondin-1(TSP-1) and signal-regulatory protein alpha (SIRPα). The referencesequence for the human mRNA has the Genbank accession numberNM_001025079, and the protein reference sequence is NP_001768.

The CD47/SIRPα interaction leads to bidirectional signaling, resultingin different cell-to-cell responses including inhibition ofphagocytosis, stimulation of cell-cell fusion, and T-cell activation

As used herein, the term “anti-CD47 agent” refers to any agent thatreduces the binding of CD47 (e.g., on an affected cell) to SIRPα (e.g.,on a phagocytic cell). In some embodiments the anti-CD47 agent does notinterfere or bind to the regions of CD47 that bind to thrombospondin. Insome embodiments, the anti-CD47 agent does not activate CD47 uponbinding. When CD47 is activated, a process akin to apoptosis (i.e.,programmed cell death) occurs (Manna and Frazier, Cancer Research, 64,1026-1036, Feb. 1 2004). Thus, in some embodiments, the anti-CD47 agentdoes not directly induce cell death of a CD47-expressing cell.

Non-limiting examples of suitable anti-CD47 reagents include highaffinity SIRPα reagents, anti-SIRPα antibodies, and anti-CD47 antibodiesor antibody fragments. In some embodiments, a suitable anti-CD47 agent(e.g. an anti-CD47 antibody, a high affinity SIRPα reagent, etc.)specifically binds CD47 to reduce the binding of CD47 to SIRPα. In someembodiments, a suitable anti-CD47 agent (e.g., an anti-SIRPα antibody,etc.) specifically binds SIRPα to reduce the binding of CD47 to SIRPα. Asuitable anti-CD47 agent that binds SIRPα does not activate SIRPα (e.g.,in the SIRPα-expressing phagocytic cell).

The efficacy of a suitable anti-CD47 agent can be assessed by assayingthe agent. An agent for use in the methods of the invention willup-regulate phagocytosis by at least 10% (e.g., at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 120%, at least 140%, at least160%, at least 160%, or at least 200%) compared to phagocytosis in theabsence of the agent. Similarly, an in vitro assay for levels oftyrosine phosphorylation of SIRPα will show a decrease inphosphorylation by at least 5% (e.g., at least 10%, at least 15%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or 100%) compared tophosphorylation observed in absence of the candidate agent.

In one embodiment of the invention, the anti-CD47 agent, or apharmaceutical composition comprising the agent, is provided in anamount effective to detectably inhibit the binding of CD47 to SIRPαpresent on the surface of phagocytic cells. The effective amount isdetermined via empirical testing routine in the art, for example in abiological sample taken from an infected individual. The effectiveamount may vary depending on the number of cells being targeted, thelocation of the cells, and factors specific to the subject.

High affinity SIRPα reagent. In some embodiments, a subject anti-CD47agent is a “high affinity SIRPα reagent”, which includes SIRPα-derivedpolypeptides and analogs thereof. High affinity SIRPα reagents aredescribed in international application PCT/US13/21937, which is herebyspecifically incorporated by reference. High affinity SIRPα reagents arevariants of the native SIRPα protein. In some embodiments, a highaffinity SIRPα reagent is soluble, where the polypeptide lacks the SIRPαtransmembrane domain and comprises at least one amino acid changerelative to the wild-type SIRPα sequence, and wherein the amino acidchange increases the affinity of the SIRPα polypeptide binding to CD47,for example by decreasing the off-rate by at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 500-fold, ormore.

A high affinity SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, e.g., high affinity,which normally lies between the signal sequence and the transmembranedomain, or a fragment thereof that retains the binding activity. Thehigh affinity SIRPα reagent will usually comprise at least the dl domainof SIRPα with modified amino acid residues to increase affinity. In someembodiments, a SIRPα variant of the present invention is a fusionprotein, e.g., fused in frame with a second polypeptide. In someembodiments, the second polypeptide is capable of increasing the size ofthe fusion protein, e.g., so that the fusion protein will not be clearedfrom the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. In otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to Fc, e.g., providing increased size,multimerization domains, and/or additional binding or interaction withIg molecules.

A suitable high affinity SIRPα reagent reduces (e.g., blocks, prevents,etc.) the interaction between the native proteins SIRPα and CD47. Theamino acid changes that provide for increased affinity are localized inthe dl domain, and thus high affinity SIRPα reagents comprise a dldomain of human SIRPα, with at least one amino acid change relative tothe wild-type sequence within the dl domain. Such a high affinity SIRPαreagent optionally comprises additional amino acid sequences, forexample antibody Fc sequences; portions of the wild-type human SIRPαprotein other than the dl domain, including without limitation residues150 to 374 of the native protein or fragments thereof, usually fragmentscontiguous with the dl domain; and the like. High affinity SIRPαreagents may be monomeric or multimeric, i.e. dimer, trimer, tetramer,etc.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding. Non-limiting examples of suitable antibodies include clonesB6H12, 5F9, 8B6, and C3 (for example as described in InternationalPatent Publication WO 2011/143624, herein specifically incorporated byreference).

Anti-SIRPα antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds SIRPα (i.e., an anti-SIRPα antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). Suitableanti-SIRPα antibodies can bind SIRPα without activating or stimulatingsignaling through SIRPα because activation of SIRPα would inhibitphagocytosis. Instead, suitable anti-SIRPα antibodies facilitate thepreferential phagocytosis of infected cells over non-infected cells.Those cells that express higher levels of CD47 (e.g., infected cells)relative to other cells (non-infected cells) will be preferentiallyphagocytosed. Thus, a suitable anti-SIRPα antibody specifically bindsSIRPα without activating/stimulating enough of a signaling response toinhibit phagocytosis.

Suitable antibodies include fully human, humanized or chimeric versionsof such antibodies. Humanized antibodies are especially useful for invivo applications in humans due to their low antigenicity. Similarlycaninized, felinized, etc antibodies are especially useful forapplications in dogs, cats, and other species respectively.

Combination Therapy: As used herein, the term “combination therapy”refers to those situations in which a subject is simultaneously exposedto two or more therapeutic regimens (e.g., two or more therapeuticagents). In some embodiments, two or more agents may be administeredsimultaneously; in some embodiments, such agents may be administeredsequentially; in some embodiments, such agents are administered inoverlapping dosing regimens.

Comparable: As used herein, the term “comparable” refers to two or moreagents, entities, situations, sets of conditions, etc., that may not beidentical to one another but that are sufficiently similar to permitcomparison there between so that conclusions may reasonably be drawnbased on differences or similarities observed. In some embodiments,comparable sets of conditions, circumstances, individuals, orpopulations are characterized by a plurality of substantially identicalfeatures and one or a small number of varied features. Those of ordinaryskill in the art will understand, in context, what degree of identity isrequired in any given circumstance for two or more such agents,entities, situations, sets of conditions, etc to be consideredcomparable. For example, those of ordinary skill in the art willappreciate that sets of circumstances, individuals, or populations arecomparable to one another when characterized by a sufficient number andtype of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder or with different sets of circumstances, individuals, orpopulations are caused by or indicative of the variation in thosefeatures that are varied.

Composition: A “composition” or a “pharmaceutical composition” accordingto this invention refers to the combination of two or more agents asdescribed herein for co-administration or administration as part of thesame regimen. It is not required in all embodiments that the combinationof agents result in physical admixture, that is, administration asseparate co-agents each of the components of the composition ispossible; however many patients or practitioners in the field may findit advantageous to prepare a composition that is an admixture of two ormore of the ingredients in a pharmaceutically acceptable carrier,diluent, or excipient, making it possible to administer the componentingredients of the combination at the same time.

Comprising: A composition or method described herein as “comprising” oneor more named elements or steps is open-ended, meaning that the namedelements or steps are essential, but other elements or steps may beadded within the scope of the composition or method. To avoid prolixity,it is also understood that any composition or method described as“comprising” (or which “comprises”) one or more named elements or stepsalso describes the corresponding, more limited composition or method“consisting essentially of” (or which “consists essentially of”) thesame named elements or steps, meaning that the composition or methodincludes the named essential elements or steps and may also includeadditional elements or steps that do not materially affect the basic andnovel characteristic(s) of the composition or method. It is alsounderstood that any composition or method described herein as“comprising” or “consisting essentially of” one or more named elementsor steps also describes the corresponding, more limited, andclosed-ended composition or method “consisting of” (or “consists of”)the named elements or steps to the exclusion of any other unnamedelement or step. In any composition or method disclosed herein, known ordisclosed equivalents of any named essential element or step may besubstituted for that element or step.

Determine: Many methodologies described herein include a step of“determining”. Those of ordinary skill in the art, reading the presentspecification, will appreciate that such “determining” can utilize or beaccomplished through use of any of a variety of techniques available tothose skilled in the art, including for example specific techniquesexplicitly referred to herein. In some embodiments, determining involvesmanipulation of a physical sample. In some embodiments, determininginvolves consideration and/or manipulation of data or information, forexample utilizing a computer or other processing unit adapted to performa relevant analysis. In some embodiments, determining involves receivingrelevant information and/or materials from a source. In someembodiments, determining involves comparing one or more features of asample or entity to a comparable reference.

Dosage Form: As used herein, the term “dosage form” refers to aphysically discrete unit of an active agent (e.g., a therapeutic ordiagnostic agent) for administration to a subject. Each unit contains apredetermined quantity of active agent. In some embodiments, suchquantity is a unit dosage amount (or a whole fraction thereof)appropriate for administration in accordance with a dosing regimen thathas been determined to correlate with a desired or beneficial outcomewhen administered to a relevant population (i.e., with a therapeuticdosing regimen). Those of ordinary skill in the art appreciate that thetotal amount of a therapeutic composition or agent administered to aparticular subject is determined by one or more attending physicians andmay involve administration of multiple dosage forms.

Dosing Regimen: As used herein, the term “dosing regimen” refers to aset of unit doses (typically more than one) that are administeredindividually to a subject, typically separated by periods of time. Insome embodiments, a given therapeutic agent has a recommended dosingregimen, which may involve one or more doses. In some embodiments, adosing regimen comprises a plurality of doses each of which areseparated from one another by a time period of the same length; in someembodiments, a dosing regimen comprises a plurality of doses and atleast two different time periods separating individual doses. In someembodiments, all doses within a dosing regimen are of the same unit doseamount. In some embodiments, different doses within a dosing regimen areof different amounts. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount different from the first dose amount. Insome embodiments, a dosing regimen comprises a first dose in a firstdose amount, followed by one or more additional doses in a second doseamount same as the first dose amount In some embodiments, a dosingregimen is correlated with a desired or beneficial outcome whenadministered across a relevant population (i.e., is a therapeutic dosingregimen).

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations. Thedefinition also includes sample that have been enriched for particulartypes of molecules, e.g., nucleic acids, polypeptides, etc.

The terms “specific binding,” “specifically binds,” and the like, referto non-covalent or covalent preferential binding to a molecule relativeto other molecules or moieties in a solution or reaction mixture (e.g.,an antibody specifically binds to a particular polypeptide or epitoperelative to other available polypeptides; high affinity binding of aSIRPα polypeptide to CD47; etc.) In some embodiments, the affinity ofone molecule for another molecule to which it specifically binds ischaracterized by a K_(D) (dissociation constant) of 10⁻⁵ M or less(e.g., 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less,10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³ M or less,10⁻¹⁴ M or less, 10⁻¹⁵ M or less, or 10⁻¹⁶ M or less). “Affinity” refersto the strength of binding, increased binding affinity being correlatedwith a lower K_(D).

The term “specific binding member” as used herein refers to a member ofa specific binding pair (i.e., two molecules, usually two differentmolecules, where one of the molecules, e.g., a first specific bindingmember, through non-covalent means specifically binds to the othermolecule, e.g., a second specific binding member). Suitable specificbinding members include agents that specifically bind CD47 (i.e.,anti-CD47 agents), or that otherwise block the interaction between CD47and SIRPα, agents that bind to calreticulin or its LRP receptor, etc.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

A “variant” polypeptide means a biologically active polypeptide asdefined below having less than 100% sequence identity with a nativesequence polypeptide. Such variants include polypeptides wherein one ormore amino acid residues are added at the N- or C-terminus of, orwithin, the native sequence; from about one to forty amino acid residuesare deleted, and optionally substituted by one or more amino acidresidues; and derivatives of the above polypeptides, wherein an aminoacid residue has been covalently modified so that the resulting producthas a non-naturally occurring amino acid. Ordinarily, a biologicallyactive variant will have an amino acid sequence having at least about90% amino acid sequence identity with a native sequence polypeptide,preferably at least about 95%, more preferably at least about 99%. Thevariant polypeptides can be naturally or non-naturally glycosylated,i.e., the polypeptide has a glycosylation pattern that differs from theglycosylation pattern found in the corresponding naturally occurringprotein. The variant polypeptides can have post-translationalmodifications not found on the natural protein.

A “fusion” polypeptide is a polypeptide comprising a polypeptide orportion (e.g., one or more domains) thereof fused or bonded toheterologous polypeptide. A fusion soluble CRT protein, for example,will share at least one biological property in common with a nativesequence soluble CRT polypeptide. Examples of fusion polypeptidesinclude immunoadhesins, as described above, which combine a portion ofthe polypeptide of interest with an immunoglobulin sequence, and epitopetagged polypeptides, which comprise a soluble polypeptide of interest orportion thereof fused to a “tag polypeptide”. The tag polypeptide hasenough residues to provide an epitope against which an antibody can bemade, yet is short enough such that it does not interfere withbiological activity of the polypeptide of interest. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 6-60 amino acid residues.

A “functional derivative” of a native sequence polypeptide is a compoundhaving a qualitative biological property in common with a nativesequence polypeptide. “Functional derivatives” include, but are notlimited to, fragments of a native sequence and derivatives of a nativesequence polypeptide and its fragments, provided that they have abiological activity in common with a corresponding native sequencepolypeptide. The term “derivative” encompasses both amino acid sequencevariants of polypeptide and covalent modifications thereof. For example,derivatives and fusion of soluble CRT find use as CRT mimetic molecules.

Small molecule: As used herein, the term “small molecule” refers toorganic compounds, whether naturally-occurring or artificially created(e.g., via chemical synthesis) that have relatively low molecular weightand that are not proteins, polypeptides, or nucleic acids. Typically,small molecules have a molecular weight of less than about 1500 g/mol.Also, small molecules typically have multiple carbon-carbon bonds.

Subject: By “subject” is meant a mammal (e.g., a human, in someembodiments including prenatal human forms). In some embodiments, asubject is suffering from a relevant disease, disorder or condition. Insome embodiments, a subject is susceptible to a disease, disorder, orcondition. In some embodiments, a subject displays one or more symptomsor characteristics of a disease, disorder or condition. In someembodiments, a subject does not display any symptom or characteristic ofa disease, disorder, or condition. In some embodiments, a subject issomeone with one or more features characteristic of susceptibility to orrisk of a disease, disorder, or condition. In some embodiments, asubject is a patient. In some embodiments, a subject is an individual towhom diagnosis and/or therapy is and/or has been administered.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. “Antibodies” (Abs) and“immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety and (4)nanobodies comprising single Ig domains from non-human species or otherspecific single-domain binding modules; and multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fcregion of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

9p21 Risk. As used herein, the term “an individual carrying at least one9p21 risk factor” refers to humans in which one or more risk alleles atthe 9p21 locus are present in the genome. Such individuals have beenshown to have an increased risk of: early onset myocardial infarction,abdominal aortic aneurysm, stroke, peripheral artery disease, andmyocardial infarction/coronary heart disease. This risk is independentof traditional risk factors, including diabetes, hypertension,cholesterol, and obesity. See, for example, Helgadottir et al. Science.2007; 316(5830):1491-1493; Helgadottir et al. Nat Genet. 2008;40(2):217-224; Palomaki et al. JAMA. 2010; 303(7):648-656; and Robertset al. Curr Opin Cardiol. 2008; 23:629-633, each herein specificallyincorporated by reference.

The 9p21 locus is in tight LD (linkage disequilibrium), and a number ofsingle nucleotide polymorphisms (SNP) markers have been shown to beuseful in diagnosis. Representative SNPs include without limitationrs10757278; rs3217992; rs4977574; rs1333049; rs10757274; rs2383206;rs2383207; Rs3217989; rs1333040; rs2383207; rs10116277; rs7044859;rs1292136; rs7865618; rs1333045; rs9632884; rs10757272; rs4977574;rs2891168; rs6475606; rs1333048; rs1333049; Rs1333045; etc.

Efferocytosis. The process by which professional and nonprofessionalphagocytes dispose of apoptotic cells in a rapid and efficient manner.Efferocytosis involves a number of molecules, including ligands on theapoptotic cells, e.g. phosphatidylserine; receptors on the efferocyte;soluble ligand-receptor bridging molecules; and so-called “find-me” and“don't-eat-me” molecules, e.g., lysophospholipids and CD47, theexpression of which by dying cells is altered to attract nearbyphagocytes. By clearing apoptotic cells at a relatively early stage ofcell death, when the cell plasma and organelle membranes are stillintact, postapoptotic, or “secondary”, necrosis is prevented. Preventionof cellular necrosis, in turn, prevents the release of potentiallydamaging intracellular molecules into the extracellular milieu,including molecules that can stimulate inflammatory, proatheroscleroticand/or autoimmune responses.

The efficiency of efferocytic clearance in atherosclerotic lesions playsa key role in disease development. Efferocytosis is known to be impairedin human atherosclerotic plaque. A prominent feature of advancedatherosclerotic lesions is the necrotic core, or lipid core, which is acollection of dead and necrotic macrophages surrounded by inflammatorycells. Necrotic cores are thought to be a major feature responsible forplaque “vulnerability”, i.e., plaques capable of undergoing disruptionand triggering acute lumenal thrombosis. Plaque disruption and acutethrombosis are the events that trigger acute coronary syndromes,including myocardial infarction, unstable angina, sudden cardiac death,and stroke.

By “manipulating efferocytosis” is meant an up-regulation or adown-regulation in efferocytosis of a targeted cell, e.g. apoptotic SMC,by at least about 10%, or up to 20%, or 50%, or 70% or 80% or up toabout 90% compared to level of efferocytosis observed in absence ofintervention.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arethree main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells. However, “non-professional” cells are also known toparticipate in efferocytosis, such as neighboring SMCs in the bloodvessel wall.

Methods

Methods are provided for treating or reducing atherosclerosis byadministering an effective combined dose of an anti-CD47 agent and ananti-TNFα agent to increase efferocytosis of cellular components ofcoronary or extracardiac plaque, including the efferocytosis ofapoptotic smooth muscle cells. Administration may be simultaneous orconcomitant, where the dosing schedule may be tailored to each of theagents in the combination. As an example, a weekly dosing of theanti-TNFα agent may be concomitant with a semi-weekly, or bi-weeklydosing of the anti-CD47 agent, or vice versa. Methods of administrationto the cardiovascular system are of particular interest.

Effective doses of the therapeutic entity of the present invention varydepending upon many different factors, including the nature of theagent, means of administration, target site, physiological state of thepatient, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human, but nonhuman mammals may also betreated, e.g. companion animals such as dogs, cats, horses, etc.,laboratory mammals such as rabbits, mice, rats, etc., and the like.Treatment dosages can be titrated to optimize safety and efficacy.

In some embodiments, the therapeutic dosage can range from about 0.0001to 500 mg/kg, and more usually 0.01 to 100 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-50 mg/kg. The dosage may be adjusted forthe molecular weight of the reagent and may be reduced relative to thedosage required for a monotherapy of either agent in the combination. Anexemplary treatment regime entails administration daily, semi-weekly,weekly, once every two weeks, once a month, etc. In another example,treatment can be given as a continuous infusion. Therapeutic entities ofthe present invention are usually administered on multiple occasions.Intervals between single dosages can be weekly, monthly or yearly.Intervals can also be irregular as indicated by measuring blood levelsof the therapeutic entity in the patient. Alternatively, therapeuticentities of the present invention can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of thepolypeptide in the patient. It will be understood by one of skill in theart that such guidelines will be adjusted for the molecular weight ofthe active agent, e.g. in the use of polypeptide fragments, in the useof antibody conjugates, in the use of high affinity SIRPα reagents, etc.The dosage may also be varied for localized administration, e.g.intranasal, inhalation, etc., or for systemic administration, e.g. i.m.,i.p., i.v., and the like.

For the treatment of disease, the appropriate dosage of the agent willdepend on the severity and course of the disease, whether the agent isadministered for preventive purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The agent is suitably administered to the patientat one time or over a series of treatments.

Suitable agents can be provided in pharmaceutical compositions suitablefor therapeutic use, e.g. for human treatment. In some embodiments,pharmaceutical compositions of the present invention include one or moretherapeutic entities of the present invention or pharmaceuticallyacceptable salts, esters or solvates thereof. In some other embodiments,the effective combined dose of an anti-CD47 agent and an anti-TNFα agentare further combined with a third therapeutic agent, e.g., drugs usefulin the treatment of atherosclerosis. Such combinations may include,without limitation, statins. Statins are inhibitors of HMG-CoA reductaseenzyme. These agents are described in detail; for example, mevastatinand related compounds as disclosed in U.S. Pat. No. 3,983,140;lovastatin (mevinolin) and related compounds as disclosed in U.S. Pat.No. 4,231,938; pravastatin and related compounds as disclosed in U.S.Pat. No. 4,346,227; simvastatin and related compounds as disclosed inU.S. Pat. Nos. 4,448,784 and 4,450,171; fluvastatin and relatedcompounds as disclosed in U.S. Pat. No. 5,354,772; atorvastatin andrelated compounds as disclosed in U.S. Pat. Nos. 4,681,893, 5,273,995and 5,969,156; and cerivastatin and related compounds as disclosed inU.S. Pat. Nos. 5,006,530 and 5,177,080. Additional agents and compoundsare disclosed in U.S. Pat. Nos. 5,208,258, 5,130,306, 5,116,870,5,049,696, RE 36,481, and RE 36,520. Statins include the salts and/orester thereof.

Therapeutic formulations comprising one or more agents of the inventionare prepared for storage by mixing the agent having the desired degreeof purity with optional physiologically acceptable carriers, excipientsor stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. The agent composition will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the agent to be administered willbe governed by such considerations, and is the minimum amount necessaryto treat or prevent atherosclerosis.

The agent can be administered by any suitable means, including topical,oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, intrathecal or subcutaneousadministration. In addition, the agent can be suitably administered bypulse infusion, particularly with declining doses of the agent.

The combination of agents may be used in the same dosages and withadministration routes as used hereinbefore as individual agents, orabout from 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85 90, 95, to 99% of the heretofore employed dosages.

An agent is often administered as a pharmaceutical compositioncomprising an active therapeutic agent and another pharmaceuticallyacceptable excipient. The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

In still some other embodiments, pharmaceutical compositions can alsoinclude large, slowly metabolized macromolecules such as proteins,polysaccharides such as chitosan, polylactic acids, polyglycolic acidsand copolymers (such as latex functionalized Sepharose™, agarose,cellulose, and the like), polymeric amino acids, amino acid copolymers,and lipid aggregates (such as oil droplets or liposomes).

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group, and non-covalentassociations. Suitable covalent-bond carriers include proteins such asalbumins, peptides, and polysaccharides such as aminodextran, each ofwhich have multiple sites for the attachment of moieties. A carrier mayalso bear an anti-CD47 agent by non-covalent associations, such asnon-covalent bonding or by encapsulation. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindinganti-CD47 agents, or will be able to ascertain such, using routineexperimentation.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Carriers and linkers specific for radionuclide agents includeradiohalogenated small molecules and chelating compounds. A radionuclidechelate may be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, or metal oxide, radionuclide.

Radiographic moieties for use as imaging moieties in the presentinvention include compounds and chelates with relatively large atoms,such as gold, iridium, technetium, barium, thallium, iodine, and theirisotopes. It is preferred that less toxic radiographic imaging moieties,such as iodine or iodine isotopes, be utilized in the methods of theinvention. Such moieties may be conjugated to the anti-CD47 agentthrough an acceptable chemical linker or chelation carrier. Positronemitting moieties for use in the present invention include ¹⁸F, whichcan be easily conjugated by a fluorination reaction with the agent.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the agents can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., bydetermining the LD₅₀ (the dose lethal to 50% of the population) or theLD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The dataobtained from these cell culture assays and animal studies can be usedin formulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition.

Cell-based systems may be used to identify compounds and combinations ofcompounds that act to ameliorate cardiovascular disease symptoms. Forexample, such cell systems may be exposed to a combination of agentscompound at a sufficient concentration and for a time sufficient toelicit such an amelioration of cardiovascular disease symptoms in theexposed cells. After exposure, the cells are examined to determinewhether one or more of the cardiovascular disease cellular phenotypeshas been altered to resemble a more normal or more wild type,non-cardiovascular disease phenotype.

In addition, animal-based disease systems, such as those described,above may be used to identify compounds capable of ameliorating diseasesymptoms. Such animal models may be used as test substrates for theidentification of drugs, pharmaceuticals, therapies, and interventions,which may be effective in treating disease. For example, animal modelsmay be exposed to a compound, suspected of exhibiting an ability toameliorate cardiovascular disease symptoms, at a sufficientconcentration and for a time sufficient to elicit such an ameliorationof disease symptoms in the exposed animals. The response of the animalsto the exposure may be monitored by assessing the reversal of disordersassociated with disease, for example, by counting the number ofatherosclerotic plaques and/or measuring their size before and aftertreatment.

With regard to intervention, any treatments that reverse any aspect ofcardiovascular disease symptoms should be considered as candidates forhuman disease therapeutic intervention. Dosages of test agents may bedetermined by deriving dose-response curves.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Example 1

It has been estimated that as many as one million cells undergoprogrammed cell death per second each day in the human body. Despite thefrequency of this event, apoptotic cells are rarely observed in vivo,even in organs with high basal turnover rates such as the bone marrow orthymus. This observation is attributed to the fact that apoptotic bodiesare rapidly and efficiently cleared by both professional (i.e.macrophage) and non-professional (i.e. neighboring cell) phagocytes.Previously considered an obligate homeostatic event, the process of‘efferocytosis’ (from the Greek: to carry the dead to the grave) is nowappreciated to occur as the result of highly orchestrated paracrinesignaling between the AB and its potential phagocyte. During programmedcell death, apoptosing cells secrete chemotactic “find-me” ligands,upregulate cell-surface “eat-me” ligands, and repress inhibitory“don't-eat-me” signals. Remarkably, this process occurs in an“immunologically silent” manner, where the successful execution of theengulfment process triggers an anti-inflammatory cytokine profile fromthe phagocyte, presumably as a signal that no further immune activationis required. Conversely, apoptotic cells which evade clearance rapidlybecome secondarily necrotic and induce an inflammatory ‘danger response’as they release toxic and antigenic intracellular content which waspreviously sequestered. Impaired efferocytosis is now recognized as amajor driver of autoimmune, inflammatory and malignant disorders, wherefailed immune surveillance is thought to result from an imbalance in thepro- and anti-phagocytosis signatures on target cells.

Atherosclerosis is a condition in which apoptosis is dramaticallyaccelerated. Complicating this is the fact that efferocytosis may bereduced by nearly ˜20-fold as the human atherosclerotic plaque develops.The reason for this defect is not clear, but is likely related in partto competition for phagocyte receptors by oxidized LDL and/or thegeneration of autoantibodies which mask important cell-surface ligandson the apoptotic body. Moreover, experimental atherosclerosis models canbe significantly accelerated by inhibiting the expression of ‘eat-me’ligands in mice, and these animals display lesions with advancednecrotic cores replete with apoptotic corpses. Impaired efferocytosislikely has important clinical consequences in atherogenesis, given thatdelayed clearance of dying SMCs has been linked to vascular inflammationand matrix destabilization, and that residual necrotic debris frequentlylocalizes to regions of the lesion most susceptible to rupture. The factthat loss of Cdkn2b in mice increases the size of the plaque and itslipid core while reducing the thickness and stability of the fibrous capmay partially explain the simultaneous link in humans between 9p21 andboth total CAD burden and acute clinical events, such as myocardialinfarction. Impaired efferocytosis may also promote atherosclerosissecondarily through the phagocyte.

Emerging evidence has revealed that the phenotype of the apoptosing cellcan have a dramatic impact on the behavior of the nearby macrophage andits ultimate capacity to maintain lipid homeostasis. Under physiologicalconditions, macrophages which have successfully engaged an apoptoticbody upregulate transmembrane export pathways downstream of LRP-1 and avariety of nuclear receptors, presumably in preparation for theimpending doubling of their intracellular content. A key effectormolecule in this pathway is ABCA1, which promotes reverse cholesterolefflux and is important for limiting the local accumulation ofcholesterol in the fatty streak. In the current study, macrophagespresented with CDKN2B-deficient apoptotic bodies failed to activate thispathway and could not process oxidized lipids efficiently.Mechanistically, this likely occurred because CDKN2B deficient cellsexpress low levels of CALR, which is a well described ligand for theLRP-1 receptor. As a consequence, these otherwise healthy macrophagesdisplayed a blunted increase in ABCA1 expression and were more likely todifferentiate into foam cells—a process which is recognized asmaladaptive and pro-atherosclerotic. Thus, while the efferocyticcapacity of the phagocyte is not altered by its basal CDKN2B expression,its ultimate participation in the atherogenesis process is highlydependent on whether it encounters a ‘normal’ AB, or one that has beenrendered ‘inedible’ due to a lack of CDKN2B.

CDKN2B mediates its phagocytic effects through calreticulin.Calreticulin is an evolutionarily conserved 46 kD chaperone proteinwhich regulates a variety of cellular functions including calciumhomeostasis, cell adhesion, wound healing, immunity, fibrosis and theresponse to stress. Additionally, CALR has been implicated as one of themajor regulators of efferocytosis, and is a critical engulfment ligandwhich is absolutely required for phagocytic clearance. During apoptosis,CALR co-localizes to the surface of the AB with exposedphosphatidylserine and activates LRP-1 on the surface of the adjacentmacrophage. Interestingly, prior studies have shown that mice deficientin this CALR receptor phenocopy several aspects of the CDKN2B-deficientmouse, including the propensity to develop large aortic aneurysms andadvanced lipid-laden atherosclerotic plaques, with no difference inplasma lipoprotein levels (reviewed in Boucher and Herz (2011) BiochemPharmacol 81:1-5). Further, CALR has also been identified as a keyregulator of tumor surveillance and the clearance of malignant cells.

Because the 9p21 locus promotes risk independently of all classical riskfactors, a therapy that promotes efferocytosis can provide incrementalbenefit beyond antihypertensives, antidiabetics and lipid loweringtherapies.

Our data demonstrates that therapies that promote phagocytosis and/orefferocytosis (including but not limited to anti-CD47 Ab therapy)dramatically reduce atherosclerosis, as shown in FIG. 1 and aneurysmdisease, as shown in FIG. 2 We have shown that such therapies reduce thenumber of apoptotic cells present in the developing vascular lesion,shown in FIG. 3, even though they have no direct effect on programmedcell death or apoptosis itself, in vitro. Using both in vitro and invivo assays (shown in FIGS. 3 and 4), we have confirmed that anti-CD47Ab therapy stimulates the phagocytosis/efferocytosis of vascular cells,which therefore accounts for the reduced number of apoptotic cells inthe atherosclerotic plaque, and consequently accounts for the reducedsize of the atherosclerotic lesion.

To understand why anti-CD47 Ab therapy is so potent in the vascularlesion, we treated vascular cells with the pro-atherosclerotic cytokine,TNF-alpha, and found that this agent blunted the expected decrease inCD47 mRNA expression that occurs during cellular stress and/or apoptosisover time (FIG. 5, STS=staurosporine). Similarly, TNF-alpha treatmentblunted the apoptosis-induced reduction in cell-surface expression ofCD47 on vascular cells (FIG. 6). Because TNF-alpha is known to beexpressed in the atherosclerotic plaque, and we now show that TNF-alphaupregulates expression of the anti-phagocytic CD47 molecule, these dataexplain why diseased vascular cells become resistant tophagocytosis/efferocytosis in vascular disease.

We confirm that CD47 expression is upregulated in human atherosclerotictissue relative to non-atherosclerotic tissue (FIG. 7) and confirm thatit is specifically upregulated within the atherosclerotic plaque inhuman lesions. Signaling studies confirm that the CD47 promoter containspredicted transcription factor binding sites for molecules known to bedownstream of TNF-alpha. Luciferase reporter assays confirm thatoverexpression of a representative TNF-alpha effector molecule, NFκB1,leads to CD47 expression in vascular cells, confirming the mechanism ofaction (FIG. 8). Microarray assays reveal that CD47 expression ispositively correlated with NFκB1 in human coronary artery samples (FIG.9, right) and TNF-alpha in human carotid plaque samples (FIG. 10, left),confirming the association in human tissue.

In vitro phagocytosis assays demonstrate that TNF-alpha impairs thephagocytosis of vascular cells at baseline and under a number of diseaseconditions including exposure to pro-atherosclerotic lipids and otherapoptosis-inducing stimuli (FIG. 11). Our data confirm that anti-CD47 Abtherapy is particularly efficacious in the presence of TNF-alpha andidentify dual inhibition of CD47 and TNF-alpha (and its relatedsignaling members) as a target for the prevention and treatment ofcardiovascular disease.

Methods:

Murine atherosclerosis studies. Animals used in this study included maleCdkn2b^(+/+), ApoE^(−/−) (n=27, Jackson Laboratory) and Cdkn2b^(−/−),ApoE^(−/−) (n=27) mice on a C57BL/6 background, which were bred by ourlaboratory as previously described. At 4 weeks of age, the animals wereweaned and initiated on a high fat Western diet (21% anhydrous milk fat,19% casein and 0.15% cholesterol, Dyets no. 101511) for the ensuingweeks. Animals were observed daily, and in the case of premature suddendeath, necropsy was performed to determine the cause of death. Lipidanalysis was performed in mice after an overnight fast, as previouslydescribed. In brief, total plasma cholesterol (CHOD-PAP; RocheDiagnostics), HDL (HDL-C-plus 2nd generation; Roche Diagnostics), andLDL concentrations (GPO-PAP; Roche Diagnostics) were measured usingenzymatic kits on an automated analyzer (Roche) according to themanufacturer's instructions. Fasting glucose was measured in venousblood from a tail prick using a Freestyle Glucometer and glucose strips(Abbott). At 16 weeks of age, the mice were euthanized and the aortaswere isolated and processed for analysis. A subset of ten mice wereimplanted with subcutaneous osmotic minipumps (Alzet, Model 2004) afteronly four weeks of high-fat diet, to deliver 1.4 mg/kg/day ofAngiotensin II for 72 hours prior to sacrifice to enhance vascularinjury in early atherosclerotic lesions. All studies were approved bythe Stanford University Administrative Panel on Laboratory Animal Careand conform to the Guide for the Care and Use of Laboratory Animalspublished by the US National Institutes of Health.

Aortic and brachiocephalic tissue preparation, immunohistochemistry andatherosclerotic lesion quantification. Aortic atherosclerosis lesionarea was determined as described previously. Briefly, the arterial treewas perfused with PBS (pH 7.3) and then perfusion fixed withphosphate-buffered paraformaldehyde (3%, pH 7.3). The heart and thefull-length of the aorta-to-iliac bifurcation was exposed and dissectedcarefully from any surrounding tissues. Aortas were then opened alongthe ventral midline and dissected free of the animal and pinned outflat, intimal side up, onto black wax. Aortic images were captured witha digital camera mounted on a Nikon stereomicroscope and analyzed usingAdobe Photoshop CS5 software. The percentage of lesion area wascalculated as total lesion area divided by total surface area. Theatherosclerotic lesions in aortic valve area and proximalbrachiocephalic artery were analyzed as described previously. Thesamples were perfused with PBS, fixed with paraformaldehyde (4%),embedded in OCT, and sectioned at 7-μM thickness. Three sections at100-μM intervals were collected from each mouse and stained with Oil RedO (Sigma-Aldrich, 00625), Masson Trichrome (Sigma Aldrich, St. Louis,Mo., USA), Picrosiruis Red (Polysciences, #24901), haematoxylin andeosin (H&E), smooth muscle α-actin (SMA, Abcam, ab5694, 1:300), Mac-3(BD Sciences BD 550292, 1:75), CD-3 (Abcam, ab5690, 1:150), andCalreticulin (Abcam, ab2907, 1:300). Biotinylated secondary antibodiesfollowed by avidin-biotin-alkaline phosphatase substrate were used aspreviously described.

In vivo apoptosis was assessed by staining for TUNEL positivity with theCell Death Detection Kit (Roche), per protocol. Cellular proliferationwas measured by staining with PCNA (Abcam, ab2426, 1:500). Thecellularity of the vessel was measured by manually counting nuclei ofsections stained with DAPI. Negative controls were performed with theomission of the primary antibody. The lesion areas were measured andquantified using Adobe Photoshop. Features of atherosclerotic plaquevulnerability were assessed as previously described. Briefly, the sizeof the necrotic core was measured by calculating the percentage of thelesion which was acellular on H&E staining. The cap thickness wasmeasured by placing a 12 point compass in the center of the blood vesseland averaging the thickness of the cap at each point as it crossed thelesion. SMC cap coverage was measured by calculating the percentage ofthe fibrous cap surface which stained positive for SMA-actin. Otherstandard features were assessed as described. Samples harvested fromseveral tissue beds were also snap frozen in liquid nitrogen forsubsequent gene expression analysis, as described below. Electronmicroscopy was performed in the Cell Sciences Imaging Facility by theStanford Electron Microscopy Core on a Jeol TEM1230.

Human atherosclerotic plaque harvest and gene coexpression networkanalysis. Details of sample collection, RNA isolation, and microarrayhybridization have been previously described. In brief, epicardialcoronary arteries were harvested by dissection from explanted hearts of22 patient donors for orthotopic heart transplant. Arterial segmentswere identified as containing atherosclerotic lesions (n=38) or not(n=13) by microscopic inspection. RNA was isolated from each sample andhybridized to a custom dual-dye gene expression microarray (Agilent;Palo Alto, Calif.) representing 20,226 transcripts identified viasequencing clones from stimulated vascular cells, literature review forgenes important to cardiovascular function, and combination with acommercial clone set (Incyte). Arrays were scanned using Agilent'sG2565AA Microarray Scanner System and Agilent feature extractionsoftware was used to generate log 2 ratios and P values for features onthe array. Prior to gene coexpression network analysis, probe setidentifiers were mapped to the current NCBI refseq gene build (hg19) andmedian values were taken for probes matching the same transcript ID. Thegeneral framework for weighted gene coexpression network analysis isdescribed.

Pair-wise Pearson correlation between gene expression values wascalculated for every gene in the dataset for a) samples withatherosclerotic lesions, b) samples without atherosclerotic lesions, c)all samples. A soft thresholding parameter β was chosen to satisfyscale-free topology criterion based on R2 maximization for a linear fitwith slope −1 to log(k) vs. log(n(k)), effectively “noising down” weakcorrelations. The topological overlap between genes was calculatedaccording to the method described by Yip and Horvath, generating anetwork adjacency based on shared network neighbors for all gene pairs.We next used average linkage hierarchical clustering and the dynamictree cut algorithm, which iteratively searches for stable clusters, topartition the topological overlap network into modules. Singular valuedecomposition was used to identify the module “eigengene” (firstprinciple component) representing the maximum variance in modular geneexpression, and the intra-modular and global connectivity for each genewas generated by summing edge weights within modules and within theglobal network, respectively.

For targeted analysis of the topological relationship between CDKN2B and28 annotated genes involved in efferocytosis (CALR, MFGEB, CX3CL1,ABCA6, ICAM3, GAS6, APOH, PROS1, C1QB, ANXA1, CD47, LRP1, MBL2, SIRPA,NR1H3, PPARG, LRPAP1, TGFB1, BAI1, TIMD4, CD14, MERTK, CD36, ELMO1,DOCK1, AKT1, PANX1, GULP1), average linkage hierarchical clustering wasperformed on the reduced topological overlap matrix representing allpair-wise links between these set members and CDKN2B. Module assignmentand eigengene calculation was performed as described above. Differentialexpression analysis according to presence or absence of atheroscleroticlesion was performed by Wilcoxon rank sum test between module eigengeneexpression values. A p value less than 0.05 was considered statisticallysignificant. Network visualization was performed using Cytoscape 2.8.3(San Diego, Calif.) to the topological overlap matrix.

Cell culture methods. Human coronary artery SMC (HCASMC, Lonza,Walkersville, Md., passage #3-6) were propagated in SmGM-2 growth media(Lonza) containing 5% FBS. Human THP-1 monocytic cells, human embryonickidney (HEK-293) and RAW 264.7 macrophages (ATCC) were grown inDMEM-growth media containing 10% FBS. Primary vascular smooth musclecells were harvested from the aortas of Cdkn2b^(+/+) and Cdkn2b^(−/−)mice, as previously described. Primary activated macrophages wereharvested from mice 72 hrs after intraperitoneal injection of 2 ml of 4%thioglycollate, as previously described. To induce growth arrest and theexpression of differentiation genes, SMC were serum starved in basalmedia (SmBM) for 72 hours, according to conventional protocols.

To induce differentiation of THP-1 monocytes into adherent macrophages,cells were treated with 100 nM PMA for 72 hours, as previouslydescribed. For knockdown experiments, SMC were transfected with 300 nMof anti-CDKN2B (siCDKN2B) or high-GC negative control (siCont) siRNA(Ambion, Silencer Select, catalog #4390825 and 4390843, respectively)using the high-efficiency Amaxa Nucleofector system (Lonza, protocolU-025). Successful transfection (>85% of all cells) was confirmed byvisual fluorescent microscopic analysis and fluorescence activated cellsorting (FACS) flow cytometry for the fluorescently-labeled positivecontrol, pmax GFP (Amaxa). Plates were harvested at 80% confluence forRNA and protein analysis or used for subsequent in vitro analysis.Reproducible knockdown of CDKN2B was confirmed in SMC by quantitativert-PCR which displayed selective silencing of this gene on the order of˜85%. No off target knockdown was observed for any of the other nearbygenes, including CDKN2A, ARF or ANRIL. Apoptosis was induced by treatingHCASMC with 1 μM staurosporine (Sigma, S5921) in serum free media for 6hours prior to analysis or harvest and use in co-culture experiments.

mRNA isolation and quantitative reverse-transcription. PCR RNA wasisolated from cell lysates using the miRNeasy Mini Kit (Qiagen,Valencia, Calif.) according to the manufacturer's protocol. RNA wasisolated from the murine organ samples using the Trizol method(Invitrogen). RNA was quantified with the Nanodrop machine (AgilentTechnologies, Santa Clara, Calif.) For quantitation of genetranscription, cDNA was generated with M-MuLV reverse transcriptase, andthen amplified on the ABI PRISM 7900HT with commercially availableTaqMan primers (Applied Biosystems, Foster City, Calif.) and normalizedto 18S internal controls, as previously described. A list of the primersand probes used in these studies is provided in below.

Primer Species: Human APOH Hs00979406_m1, C1QC Hs00757779_m1, ANRILHs01390879_m1, ARF Hs99999189_m1, CDKN2A Hs00923894_m1, CDKN2BHs00793225_m1, CD47 Hs00179953_m1, CALR Hs00189032_m1, GAS6Hs01090305_m1, ICAM3 Hs00233674_m1, MFGE8 Hs00170712_m1, MTAPHs00559618_m1.

Species: Mouse Calreticulin Mm00482936_m1, Cdkn2a Mm00494449_m1, ArfMm01257348_m1, Cdkn2b Mm00483241_m1, Abca1 Mm00442646_m1.

In vitro assays and promoter analysis, In silico bioinformatics.Transcription factor binding site (TFBS) prediction was determined usingthe following online bioinformatics tools: TRANSFAC (BIOBASE), TFSearch,PROMO, and MatInspector.

Radioactive electrophoretic gel mobility shift assays. Double strandedoligonucleotides for the top predicted E2F4 binding sites (−150 to −134)within the CALR promoter were generated by annealing the followingsingle stranded oligos: F:5′ TGGCAGGGGCGGGCCCAAGGGCTG 3′ and R:5′CAGCCCTTGGGCCCGCCCCTGCCA 3′. -ATP (Perkin Elmer) using T4 polynucleotidekinase (NEB) for 30 minutes at room temperature and then purifiedthrough Sephadex G-50 Quick Spin columns (Roche). After measuringradioactivity, reactions were assembled with 1×EMSA binding buffer, 1 μgpoly-dIdC, 10 μg nuclear extract harvested from HCASMC, 100× unlabeledprobe (for competitions), -ATP labeled probe, and incubated at roomtemperature for 30 min prior to protein separation on a 4% TBE gel. Gelswere dried on Whatman paper using a heated vacuum drier and proteinswere detected on radiographic film.

Chromatin immunoprecipitation assays Chromatin immunoprecipitation(ChIP) was performed according to the Millipore EZ-ChIP protocol withslight modifications. HCASMC were cultured in normal growth media untilapproximately 75% confluent, and then cultured in the absence of serumand supplements for 24 hours. Cells were fixed in 1% formaldehyde for 10minutes to cross-link chromatin, followed by quenching with glycine for5 minutes at room temperature. 2×10{circumflex over ( )}7 cells percondition were collected, and nuclear lysates were prepared as describedpreviously (70). Cross-linked chromatin nuclear extracts were shearedinto approximately 500 bp fragments using a Bioruptor (Diagenode) for 3cycles of 3 minutes (30s ON, 30s OFF). Sheared chromatin was clarifiedvia centrifugation at 4 C for 15 minutes. 1×10{circumflex over ( )}6nuclei per condition was precleared with 20 μg anti-rabbit IgGpre-immune serum (Sigma) and 40 μl Protein G Dynabeads (Invitrogen) for1 hour on a rotating platform, followed by incubation with 2 μg RabbitIgG or anti-E2F4 antibody (C-20 SC866 Santa Cruz) overnight.

Immunoprecipitated chromatin samples were then incubated with 60p1Protein G Dynabeads for 2 hours at 4 C on a rotating platform to capturethe protein-DNA complexes. Complexes were washed in Low salt, High salt,LiCl, and TE buffers and then eluted with a buffer containing 100 nMNaHCO₃ and 1% SDS. Protein-DNA crosslinks were reversed and samples weretreated with RNase A and Proteinase K and free DNA was purified usingQiagen PCR purification kits. Total enrichment was measured usingprimers designed based on the sequence of the top E2F4 binding sitewithin the Calreticulin promoter (F (−199 to −181):5′AGGTCCAATGGAAAAAGAC 3′ and R (+84 to +65):5′ CAGAAACTGCTCCTTGAAGT 3′),or a known E2F4 regulatory region (FGFr1 as a positive control (DevelDynamics 2005 119), or a Negative Control region using the followingprimers (F:5′ CCGGAAGCACTTCTCCTAGA 3′ and R:5′ AAGAGAGAGCGGAAGTGACG 3′).

Semiquantitative PCR was used to verify ChIP products via gelelectrophoresis. Quantitative real-time PCR (ViiA 7, Life Technologies)was performed using SYBR Green (Applied Biosystems) assays and foldenrichment was calculated by measuring the delta Ct−delta Ct IgG.Melting curve analysis was also performed for each ChIP primer. Data arerepresentative of at least four independent HCASMC samples with qPCRassays performed in triplicate. Data is presented as the percentage ofInput DNA and as fold enrichment of chromatin precipitated with the E2F4Ab relative to the control IgG.

Luciferase promoter reporter assays. Calreticulin LightSwitch PromoterReporter GoClones (RenSP, S721464), Empty vectors (S790005) andCypridina TK Control constructs (pTK-Cluc, SN0322S) were obtained fromSwitchGear Genomics (Menlo Park, Calif.) and transfected into HEK cellsusing Lipofectamine 2000 (Invitrogen). For knockdown assays, 5 pmol ofanti-CDKN2B or control siRNA were co-transfected. For overexpression,CDKN2B (sc319536) and Rb expression plasmid (sc119971) and empty vector(pCMV6) were obtained from Origene and 100 ng of plasmid wereco-transfected. Dual luciferase activity was measured with theLightSwitch Dual Assay System after 48 hours using a SpectraMax Lluminometer (Molecular Devices), according to the manufacturer'sinstructions. In some experiments, media was changed to serum-free mediaafter 24 hours of transfection. Studies were performed at baseline, andafter the cells had been exposed to escalating doses of recombinanthuman TGFβ-1 (from Sigma, 0.5-10 ng/mL) for the final 16 hours prior toanalysis. Relative luciferase activity (Renilla/Cypridina luciferaseratio) is expressed as the percentage change relative to the basalvalues obtained from control-transfected cells not exposed to TGF-βtreatment.

Efferocytosis resistance and capacity assays. Primary aortic smoothmuscle cells generated from Cdkn2b^(−/−) and Cdkn2b^(+/+) mice werelabeled with 20 μM orange CMTMR CellTracker fluorescent probes (LifeTechnologies, C2927) for one hour, then cultured overnight in serum freemedia. Simultaneously, primary intraperitoneal Cdkn2b^(+/+) macrophageswere labeled with 20 μM of green CMFDA CellTracker probe (LifeTechnologies, C7025) for one hour, then cultured overnight in standardmedia with serum supplementation. In the morning, the SMCs were inducedto undergo apoptosis for 4 hours, then were harvested and manuallycounted. 1×10⁵ apoptotic cells were then added to the culturedmacrophages and were allowed to co-culture for an additional 1.5 hrs. Atthat point, all adherent cells were trypsinized and FACS sorted (BDFACSCaliber, 530 nm [FL1] and >575 nm [FL4]), as in previously publishedprotocols. Cells which were dual-positive for green (phagocyte) andorange (SMC) were assumed to represent phagocytosed cells.

The efferocytosis rate was then defined as the percentage of dualpositive cells (phagocytosed AB) to orange-positive/green-negative cells(un-eaten AB). Comparison was made between the rates of clearance forCdkn2b^(−/−) and Cdkn2b^(+/+) AB. This experiment was performed as abovewith the following permutations: primary murine aortic SMC vs. primarymurine intraperitoneal thioglycollate stimulated macrophages; primarymurine aortic SMC vs. murine unstimulated RAW macrophages; transfectedHCASMC vs. human PMA-stimulated THP-1 cells.

Finally, the effect of CDKN2B on the ‘efferocytic capacity’ (vs‘efferocytic resistance’) was assessed by performing these experimentswith CDKN2B deficient and control transfected phagocytes exposed tountransfected AB. In these experiments, the phagocytic capacity wasdefined as the percentage of dual positive cells (phagocytes which hadeaten an AB) to orange-negative/green-positive cells (phagocytes thathad not eaten an AB). All assays were repeated on three occasions withat least three replicates per experiment. Analysis was performed withFloJo 7.6.3.

Efferocytosis competition assays. Confirmation of the preceding studieswas performed by plating equal numbers of green CellTracker labeledapoptotic siCDKN2B HCASMC and orange CellTracker labeled apoptoticsiCont HCASMC onto unlabeled untransfected non-apoptotic HCASMC in12-well cell culture plates. All three cell types were co-cultured foran additional 2 hours, and then the non-adherent, non-phagocytosed cellswere washed off. The remaining cells were fixed and stained with DAPIand analyzed under an inverted fluorescent microscope. 8 random hpf/wellwere manually counted by a blinded investigator for efferocytosed cellsand the ratio of phagocytosed CDKN2B-deficient AB to control-transfectedAB was recorded.

Phagocyte-Apoptotic Body co-culture assays Cholesterol efflux cultureassays Cholesterol efflux assays were performed as described previously,with modification. RAW macrophages were plated on 12-well plates in DMEMcontaining 10% FBS and labeled with [3H]cholesterol (0.5 μCi/well) for48 hours. After washing with PBS, the cells were co-cultured withapoptotic Cdkn2b^(−/−) and Cdkn2b^(+/+) aortic smooth muscle cells for1.5 hours, then incubated in serum free DMEM overnight. The cells werewashed and incubated in 350 μl of serum free media containing 10 μg/mlApolipoprotein A-1 (Sigma) as an acceptor for 4 hours. The media wascollected and centrifuged, and the amount of radioactivity wasdetermined by scintillation counter. Cholesterol efflux was expressed asthe percentage of counts in the media versus total [³H] cholesterolcounts (media plus cell). Baseline efflux (without apoA-1) wassubtracted.

Foam cell formation assays. RAW macrophages were seeded on 96-wellplates and cultured overnight. In some experiments, macrophages weretreated with 100 ng/ml of Lipopolysaccharide from Escherichia coli0111:134 (LPS, Sigma). Macrophages were co-cultured with apoptoticCdkn2b^(−/−) and Cdkn2b^(+/+) aortic smooth muscle cells and 100 μg/mloxidized LDL (Biomedical Technologies Inc.) for 24 hrs. The cells werefixed in 4% paraformaldehyde for 20 min, washed with PBS, and stainedwith 0.5% oil red O for 5 min. After rinsing in 60% isopropanol andwashing, 8 random images/well were taken with an inverted microscope at20× magnification. Oil red O positive area was analyzed with AdobePhotoshop CS5 software.

Macrophage-specific cytokine expression assays LPS-stimulated (1 μg/mL)RAW macrophages were co-cultured with either siCDKN2B or siContapoptotic HCASMC in serum free media. Unattached HCASMC was removed bywashing with PBS after 1.5 hours, then the cells were cultured in serumfree DMEM. After 24 hours of incubation, the supernatant was collectedand the level of secreted IL-10 and TNF-α was assessed with ELISAs (R&DSystems) developed specifically for cytokines of murine origin.

Statistical analysis Data are presented as mean±SEM. Data were subjectedto the Kolmogorov-Smirnov test to determine distribution. Groups werecompared using the Mann-Whitney U test for non-parametric data or theStudents t-test for parametric data. When comparing multiple groups,data were analyzed by analysis of variance with Bonferroni's post test.For multiple testing of parametric data, a value of P<0.05 wasconsidered statistically significant. Experiments were replicated atleast in quadruplicate and all analyses were performed in a blindedfashion by two separate investigators, unless otherwise specified.Statistical analysis was performed with GraphPad Prism 5.

What is claimed is:
 1. A method of treating a subject foratherosclerosis, the method comprising: administering to the subject aneffective dose of an anti-CD47 agent and an anti-TNFα agent.
 2. Themethod of claim 1, wherein the subject is a mammal.
 3. The method ofclaim 2, wherein the subject is a human.
 4. The method of claim 1,wherein the anti-CD47 agent reduces the binding of CD47 on an apoptoticcell to SIRPα on a phagocytic cell.
 5. The method of claim 4, whereinthe anti-CD47 agent specifically binds CD47.
 6. The method of claim 5,wherein the anti-CD47 agent is an antibody.
 7. The method of claim 6,wherein the antibody is humanized 5F9-hIgG4.
 8. The method of claim 4,wherein the anti-CD47 agent is a soluble SIRPα reagent.
 9. The method ofclaim 8, wherein the agent is a high affinity soluble SIRPα reagent. 10.The method of claim 4, wherein the anti-CD47 agent specifically bindsSIRPα.
 11. The method of any one of claims 1-10, wherein the anti-TNFαagent is an antibody that binds to TNFα.
 12. The method of any one ofclaims 1-10, wherein the anti-TNFα agent is a soluble TNF receptor. 13.The method of any one of claims 1-12, wherein the combination of agentsprovides for a synergistic effect relative to the use of either agent asa monotherapy.
 14. The method of any one of claims 1-13, wherein thedosage of one or both agents in the combination is reduced relative tothe effective dose of the agent as a monotherapy.
 15. The method ofclaim 1, wherein the subject has been diagnosed as having at least one9p21 risk allele for atherosclerosis.